This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The cytochrome P450 enzymes were first isolated and reported in 1966 and have subsequently been found to be ubiquitous in nature. In Man, there are about 60 P450 enzymes, involved mainly in oxidation reactions. P450s in the liver are broad spectrum oxidants that metabolize drugs, pro- drugs, and xenobiotics, making them the most important enzymes from a pharmacological perspective because they control drug dosing and clearance. Other P450s are intimately involved in disease states such as breast cancer (due to over-production of the P450 enzyme aromatase that makes estradiol), prostate cancer (linked to over-production of P450 CYP17), and liver disease (due to over-production of the inducible P450 CYP2E1). Physical, kinetic and mechanistic studies of P450 oxidants will provide valuable information, but those studies have been precluded by the inability to produce the oxidizing intermediates at high conversion under controlled conditions. P450s are heme-containing enzymes that differ from other heme enzymes in that they have a thiolate (from protein cysteine) as the fifth ligand to iron. They have long been thought to effect oxidation by reaction of an iron(IV)-oxo porphyrin radical cation known as Compound I, which is related to well characterized Compound I derivatives of other heme-containing enzymes such as peroxidases and catalases. The one electron reduced species from Compound I, an iron(IV)-oxo species with a neutral porphyrin, is termed Compound II. Despite the common belief that Compound I is the active oxidant of a P450, these species have not been available from rapid stopped-flow mixing or rapid freeze quench studies that date back to 1968. In fact, the improvements in mixing methods in the past four decades have been marginal, and progress in detecting P450 oxidants has been slow despite dozens of reported unsuccessful or marginally successful attempts and, one assumes, scores of unreported failures. Our group developed a new approach for production of the iron-oxo derivatives of P450 enzyme